Gliederung

Introduction: Microparticles (MPs) are small particles (<1Âµm) and usually released into circulation upon activation or apoptosis of vascular cells. The content of MPs (e.g. proteins, RNA) can be transferred to target cells by membrane fusion. Recently, MPs have been reported to harbor activated caspase enzymes. So far, there are no reports about biological effects of MPs on malignant cells. The aim of this study was therefore, to investigate the effects of circulating MPs, as found in the blood of healthy volunteers, on a malignant esophageal carcinoma cell-line.

Materials and methods: Circulating MPs were isolated from blood samples of healthy volunteers (n=20) and prepared using a centrifugation protocol followed by FACS guided characterization. Kyse-270 squamous epithelial esophageal carcinoma cells were incubated with various concentrations of MP (1 - 20.000 MPs/Âµl) for 48h followed by FACS analysis (Annexin V/Propidiumiodide) to determine viable, apoptotic and necrotic cells. To evaluate caspase dependent apoptosis, either MP or Kyse cells were pre-incubated with the pan-caspase inhibitor zVAD followed by FACS analysis. Furthermore expression and activity of caspase-3 in MP preparations was defined by westernblot/caspase-3 assay.

Results: Incubation of Kyse cells with MPs for 48 h led to concentration-dependent induction of apoptosis with significant reduction of viable cells leading to 71,4% (20.000 MP/Âµl) vs. 93,9% (control). Apoptosis induction could be prevented by pre-incubation of Kyse cells with z-VAD as well as by pre-incubation of MP with z-VAD. As expected, Westernblot and caspase assay revealed significant caspase-3 expression and activity indicating a functional apoptotic potency of MP.

Conclusion: We could show for the first time, that isolated MPs obtained from human blood induce concentration-dependent apoptosis carcinoma cells in vitro. Apoptosis could be prevented by pretreatment of MPs with the global caspase inhibitor zVAD. We hypothesize that MPs induce apoptosis by transferring caspases into target cells – requiring a target cell-MP interaction.